Atomic force microscope measuring device

ABSTRACT

Atomic force microscope measuring device comprising a micro-cantilever and an intensity modulated laser exciting the cantilever, wherein the measuring device comprises an optical microscope, in particular a fluorescence microscope, a confocal microscope, a fluorescence energy transfer (FRET) microscope, a DIC and/or phase contrast microscope, all of those in particular construed as an inverted microscope.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 National Stage application of International Application No. PCT/EP2015/000349 tiled on Feb. 17, 2015, which claims priority of European Patent (EP) application Serial Number 14000560.4 filed on Feb. 17, 2014 and European Patent (EP) application Serial Number 14004244.1 filed on Dec. 16, 2014, all of which are incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

Field of the Invention

The invention relates to an atomic force microscope measuring device comprising a micro-cantilever and an intensity modulated light source exciting the cantilever.

Description of the Prior Art

The invention proposed herein is based on Atomic Force Microscopy technology. It allows to determine topography, physical, chemical and biological properties of biological systems such as single cells, tissues, single molecules, protein crystal structures, over time and in physiological relevant environment. In addition this device can be applied to characterize any other type of material and it is able to work in other environments such as air ambient conditions or vacuum. Furthermore our device is useful to study cell-cell or cell-tissue interactions. This invention has been developed to be fully compatible with optical techniques as fluorescence, Differential Interference Contrast (DIC), phase contrast, Raman spectroscopy and related techniques.

The atomic force microscopy (AFM) has not stopped developing since its invention. In U.S. Pat. No. 6,330,824 B1 new imaging method for AFM is described. As a novelty this method excites the cantilever probe of the AFM using an intensity modulated laser.

There are different architectures to build an AFM based on photothermal excitation. In many cases, an optical microscope is used to focus the intensity modulated laser on the cantilever. This microscope can be simultaneously used to focus a second laser on the cantilever in order to read out its movement as. Of course, the optical microscope used in this configuration enables to see the cantilever and therefore to position and focus the laser or lasers on the cantilever. However, this configuration does not allow to use the optical microscope to obtain any other type of information from the sample such as Differential Interference Contrast (DIC), epifluorescence or similar techniques.

Other architectures use an additional set of optical elements to focus the laser for the photothermal excitation allowing to use a top-view optical microscope [5] with many restrictions. The optical microscope in this configuration is mainly used to locate properly the laser on the cantilever. However, as in the previous eases, this type of architecture is not compatible with important optical techniques for cell biology as for instance DIC, fluorescence.

Atomic Force Microscopy (AFM) has reached an exciting stage that allows studying topography, physical, chemical and biological properties and interactions of samples with piconewton sensitivity and subnanometer resolution. These capabilities together with its ability to work in liquids make AFM a powerful multifunctional toolbox to study a big variety of biological systems, ranging from single molecules to single cells or tissues, in or close to their physiological environment.

Despite of AFM is able to extract important information when working in liquids, its performance is severely hampered due to undesired hydrodynamic effects that can perturb the cantilever, limiting importantly the conditions that can be applied in order to extract quantitative information from samples. The origin of this limitation is related with the fact that many operation AFM modes require to excite the cantilever, which in the vast majority of the instruments is done by using a dither piezo. This excitation method is very easy to implement and requires a very small cost. In addition it works very efficiently in vacuum or air ambient conditions. However its performance in liquids drops dramatically since it does not shake only the cantilever but the liquid where the sample and cantilever are submerged in, which disturbs significantly the movement of the cantilever making very difficult its interpretation and hence extracting quantitative information from the sample.

The previous limitations can be overcome by exciting the cantilever using an intensity modulated laser (photothermal excitation). This laser is generally focused at the base of the cantilever and produces a very localized heating that stresses the cantilever surface resulting in a cantilever bending. Since just the cantilever is excited, its movement follows an expected behavior which is very close to an ideal driven and damped harmonic oscillator. Nevertheless, AFM's based on this excitation method has not been developed so far to be compatible with optical techniques such as DIC, fluorescence and other related techniques, lacking the possibility of addressing important questions that required both types of information to be acquired.

It is an object of the invention to improve these known devices.

SUMMARY OF THE PRESENT INVENTION

According to a first aspect of the invention, a solution to this technical problem is solved by providing an atomic force microscope measuring device comprising a micro-cantilever and an intensity modulated light source exciting the cantilever, wherein the measuring device comprises an optical microscope, in particular a fluorescence microscope, a confocal microscope, a fluorescence energy transfer (FRET) microscope, a DIC and/or phase contrast microscope, all of those in particular construed as an inverted microscope. The light source can be any source of electromagnetic radiation with a wavelength preferably within the range of 350 nm to 750 nm. The electromagnetic radiation can be coherent or non-coherent, monochromatic or non-monocromatic. In particular the intensity modulated light source can be a laser.

Due to the importance of being able to simultaneously track the topography and physical, chemical and biological properties of biological systems together with its functional state, the AFM is able to work based on photo thermal excitation but in addition is fully compatible with modem optical microscopy and spectroscopy techniques such as fluorescence, fluorescence energy transfer (FRET), confocal microscopy, DIC and/or phase contrast or Raman spectroscopy, and in general all those based on an inverted microscope. Therefore the device enables to extract and correlate the most accurate information obtained by this combination of tools.

Although there are Atomic Force Microscopes (AFM) that oscillate the cantilever by photothermal excitation, those systems are not compatible with modem optical techniques such as DIC, fluorescence microscopy, etc. A typical reason is that, in general, those AFM systems use an optical microscope to focus an intensity modulated laser on the cantilever in order to excite (oscillate) the cantilever, which eliminates the possibility of using that microscope for fluorescence imaging. On the contrary, the device described herein is designed in such a way that the intensity modulated laser can be positioned and focused on the cantilever with a special set of optical elements and nanopositioners. The optical pathway allows optical access from the top and bottom of the device without compromising the cantilever excitation mechanism. Additionally the reverse is also true, meaning that the cantilever excitation do not compromise the acquisition of information by modem optical techniques, being both types of systems able to work simultaneously. This fact together with the transparency of the cantilever, cantilever holder and sample holder is designed to be fully compatible with modem optical techniques. It is important to note that the device described herein shares some features with systems used for near field optics, however there are major differences. For instance, some systems used for near field optics, have a cantilever and attached to it a waveguide. A laser is focused on the waveguide which has to be very close to a sample (typically several nanometers or tens of nanometers) containing metals in order to excite plasmons. The device described herein does not have a waveguide attached to the cantilever. In addition, the device described herein does not use a laser to induce effects of near field optics such as the excitation of plasmons but to deflect the cantilever over time. Furthermore, the device described herein is able to extract the information from the sample without the need of containing metals. Moreover, the device utilizes a second laser which is located near the free end of the cantilever in order to read out the deflection of the cantilever. However, this second laser is not compulsory and other systems can be applied such as piezoelectric cantilevers, piezoresistive cantilevers, Doppler interferometers, etc.

Another inventive aspect relates to an atomic force microscopy measuring device, wherein the cantilever is transparent for a wave length of the visual spectrum. The cantilever can be partially or totally transparent for the wave length of the visual spectrum. This allows for a combination with important optical techniques in cell biology.

Preferably, the part of the cantilever that is close to its free end is partially transparent for at least some wave length of the visual spectrum and it is not smaller than 10 μm×10 μm. This window of transparency or partial transparency for at least some wavelength of the visual spectrum is a requirement for the device to be compatible with techniques such as DIC (Differential Interference Contrast) or equivalents techniques, which allow obtaining crucial. information about the morphology of the biological sample even when the sample is being probed by the cantilever or when there is a biological system sitting on or close to the free end of the cantilever. These types of techniques, require a special type of illumination, which has to go through the sample and be collected using a special optical microscope located opposite to the illumination source. In addition to the DIC information, the objective allows our device to get fluorescence information from the sample. However the transparency or partial transparency of the cantilever is not a requirement for the fluorescence information, since the illumination to excite the fluorescence of the sample can be driven through the objective, which collects simultaneously the fluorescence light emitted from the sample.

Preferably the cantilever has a length in the range of 10 μm to 1000 μm and/or a resonance frequency for the fundamental mode in the range of 1 Hz to 10 MHz in air ambient conditions. Finally the cantilever preferably has an oscillation amplitude when excited at the fundamental resonance in the range of 0.01 nm to 500 nm, preferably smaller than 100 nm. To optimize the measuring device it is possible to choose or adapt a cantilever by adapting the cantilever dimensions for different applications.

Preferably the wave length of the exciting light is in a range from 350 nm to 750 nm, it may be between 350 nm and 550 nm. Specifically a wave length of 405 nm may be used which has the additional advantage that it is far enough from many of the wave lengths used in fluorescence microscopy. However, depending on the cantilever material other wave lengths may be used as well. In particular, the wave length of the exciting light is chosen such that it is strongly absorbed by the cantilever.

Advantageously the position, diameter and focus of the exiting light spot on the cantilever may be adjusted and may be located at the base of the cantilever or close to the base of the cantilever. This maximizes the excitation efficiency. The exiting light spot may be smaller than 100 μm, preferably smaller than 50 μm in diameter, preferably smaller than 10 μm in diameter. This size allows exciting the cantilever with high efficiency and at the same time allows having the laser far from the biological system to avoid interactions with the sample.

There are two ways to include the scanner or scanners into the system:

-   -   1.—The scanner holds and moves the cantilever or cantilever         holder with respect the sample. In addition, if necessary, the         scanner holds and moves other elements of the device to ensure         that the excitation of the cantilever and the read out of its         movement are properly done. Equivalently and for simplicity, the         scanner can hold and move the whole device with respect the         sample. We refer to this system as head scanner.     -   2.—The scanner holds and moves the sample with respect to the         cantilever. We refer to this system as sample scanner.

Supplementary, any combination of the two previous strategies can be used in order to achieve a specific performance of the instrument.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described in more detail hereinafter with reference to an exemplary embodiment. In the drawing,:

FIG. 1 shows a schematic sketch of a head scanner,

FIG. 2 shows schematic three-dimensional cross sectional drawing o a head scanner,

FIG. 3 shows schematic three-dimensional top view drawing of a head scanner,

FIG. 4 shows a sketch of a sample scanner,

FIG. 5 shows schematic three-dimensional cross sectional drawing of a sample scanner,

FIG. 6 shows schematic three-dimensional top view drawing of a sample scanner and

FIG. 7 shows schematic two-dimensional top view drawing of a head and sample scanner (since they are identical in this perspective) with arrows that indicate the directions of movement.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

As can be seen the FIGS. 1 and 4, a 405 nm laser (7) is focused on the base of a microcantilever using a set of optical elements represented as (5) and the dichroic mirror (4) in order to photothermally excite the cantilever. The movement of the cantilever is detected using a beam deflection scheme but other methods can be also applied. The laser beam (8) is focused at the end of the cantilever using a set of optical elements represented as (9) and the dichroic mirror (3). The laser beam (8) reflects partially on the cantilever (20) and hits a gold mirror (12) that addresses the beam into a four quadrants photodetector (13) to be analyzed. Thanks to the optical properties of the dichroic mirrors (3) and (4) together with the cantilever holder (19), the cantilever itself which is totally or partially transparent and the sample holder (18), special light generated in (2) can go through the sample and be collected with an inverted microscope (21) for DIC imaging or similar techniques. In addition, the microscope (21) allows to illuminate the sample with suitable light to excite fluorescent and collect back the fluorescent emitted light from the sample according to an inverted microscope set-up. An XYZ scanner enables to locate the cantilever in different positions of the sample by either moving the sample holder (18) or the box (11) with subnanometer resolution. The actuators (15) carry out a coarse vertical movement to approach or withdraw the cantilever to the sample. In addition these actuators can tilt the box (11) with respect to the sample holder (18).

In order to ensure a very high stability of the excitation of the cantilever, the exciting laser diode is kept at a fixed temperature by using a peltier element. This thermoelectric system ensures that wavelength and intensity of the laser will not shift due to temperature changes. By modulating the current that feeds the diode, the intensity of the laser can be modulated at a certain frequency. However other ways to modulate the light intensity can be applied as for instance by using acousto-optical or electro-optical modulators. The light emitted by the laser diode is then introduced into an optical fiber in order to reach an optical collimator which is located in the head of the instrument. The optical collimator is focusable and allows to get a laser beam with parallel rays. However, in order to modify the laser waist (minimum spot diameter) the collimator can be set to produce non parallel rays as well. At the end of the collimator and attached to it there is a micro focus optics which ensures that the laser light will be focus at a certain distance, usually the distance or close to the distance the cantilever is at.

The end of the optical fiber, together with the collimator and the micro focus optics (46 in FIG. 2) are mounted on a nanopositioner system (40 in FIG. 3) which is able to travel several millimeters with nanometric resolution. This nanopositioner system allows to modify the distance between the cantilever (51 in FIG. 2) and the optical system (46). Doing so, the system has a very precise and comfortable way to modify the laser spot size on the cantilever, just by changing the position where the beam waist is. This adjustment can be very important in order to optimize the cantilever excitation for the following reasons:

-   -   1.—It enables to find the optimum laser spot diameter to excite         the cantilever using the minimum power. This fact, for instance,         can be very important when a biological sample is sitting close         the end of the cantilever, since that part of the cantilever         might be required to be at a physiological temperature and hence         to excite the cantilever with very low power can be a need.     -   2. It brings the possibility of modifying the length and/or         material of the cantilever holder (50 in FIG. 2) making the         system very versatile to work using for instance petri dishes of         different heights or dealing with a special sample holder in         order to keep the sample under incubation conditions by using         for instance air with 5% CO2 and high humidity.

In addition the nanopositioner system (40 in FIG. 3) is held by a set of nanopositioners (41 in FIG. 3) which enable to move in XY directions the optical system (46) in order to ensure that the laser hits the cantilever on the desired location. This set of nanopositioners hold as well a dichroic mirror (39 in FIG. 2) which deviates in 90 degrees the laser light coming from the optical system (46) but not other wavelengths of the visual spectrum. This way, the laser (44) can hit on the cantilever with almost normal incidence and simultaneously other types of illumination such as DIC, phase contrast or related techniques can be utilized from the top.

A laser diode can be used in the device as a source of light to excite the cantilever, however the use of laser light is not a requirement and other types of light can be used.

The advantage of our invention over the state of the art is that our device allows using a photothermal based AFM in filly combination with modern optical techniques such as DIC, fluorescence, FRET, confocal microscopy and in general all those based on an inverted microscope.

Reference numbers for FIGS. 1 and 4:

-   -   1. Atomic force microscope measuring device     -   2. DIC, phase contrast or other type of illumination to         characterize the morphology of biological samples     -   3. Dichroic mirror that reflects the 850 nm wavelength allowing         other wavelengths in the visual spectrum to go through     -   4. Dichroic minor that reflects the 405 nm wavelength allowing         other wavelengths in the visual spectrum and the 850 nm         wavelength to go through     -   5. Optical system to correct and focus the 405 nm laser beam (6)     -   6. 405 nm laser beam     -   7. 405 nm laser diode and optical fiber to drive the laser into         the optical system (5)     -   8. 850 nm laser beam     -   9. Optical system to correct and focus the 850 nm laser (8)     -   10. 850 nm laser diode and optical fiber to drive the laser into         the optical system (9)     -   11. Base of the box to hold the apparatus     -   12. Mirror that reflects the 850 nm laser (8) to hit the         photodiode (13)     -   13. Four quadrant photodiode     -   14. Band-pass filter center at 850 nm. It allows the 850 nm         laser beam to reach the photodiode (13) but blocks other         wavelengths     -   15. Actuators to move the box and hence the cantilever along the         vertical direction. These actuators for instance allow         approaching and withdrawing the cantilever to the sample     -   16. Platform that holds the box (11). The platform contains XY         positioner that allow to do a coarse movement of the box (11)         with respect to the sample holder (18)     -   17. Hollow scanner system that holds the sample holder (18). It         allows the move the sample holder (18) in three orthogonal         directions (X, Y and Z) with respect to the box (11). The system         contains in addition XY positioners that allow to do a coarse         movement of the sample holder (18) with respect to the box (11)     -   18. Sample holder     -   19. Cantilever holder     -   20. Cantilever chip     -   21. Inverted microscope. It is compatible with modern optical         techniques such as fluorescence, DIC, phase contrast, confocal         microscopy, FRET, raman spectroscopy     -   22. Hollow scanner system that allows to move the box (11) with         respect to the sample holder (18) in three orthogonal directions         (X, Y and Z). In addition the scanner contains two positioners         to do a coarse movement of the box (11) with respect to the         sample holder (18) Reference numbers for FIGS. 2 to 3 and 5 to         7:     -   30. Atomic force microscope measuring device     -   31. Base of the box to hold the apparatus     -   32. Actuators to move the box and hence the cantilever along the         vertical direction. These actuators for instance allow         approaching and withdrawing the cantilever to the sample     -   33. Actuator that moves the mirror (43) by spinning the minor's         holder. This actuator allows addressing (reflecting) the 850 nm         laser (45) on the photodiode (49)     -   34. Dichroic mirror that reflects the 850 nm wavelength allowing         other wavelengths in the visual spectrum to go through     -   35. Positioner system that holds the optical system to focus the         850 nm laser (45). This system can move as shown by the arrows,         which for instance allows modifying the laser spot diameter on         the cantilever (51)     -   36. Positioner system that holds the positioner system (35) and         the dichroic mirror (34). The system can move as shown by the         arrows, which allows positioning the 850 nm laser beam (45) to         hit on a certain place as for instance the cantilever (51)     -   37. Window to allow DIC, phase contrast or other illumination         types     -   38. Positioner system that allows moving the photodiode as shown         by the arrows. This system for instance can be used in         combination with (33) to make the 850 nm laser hit on a certain         part of the photodiode     -   39. Dichroic mirror that reflects the 405 nm wavelength allowing         other wavelengths in the visual spectrum and the 850 nm         wavelength to go through     -   40. Positioner system that holds the optical system to focus the         405 nm laser (44). This system can move as shown by the arrows,         which for instance allows modifying the laser spot diameter on         the cantilever (51)     -   41. Positioner system that holds the positioner system (40) and         the dichroic mirror (39). The system can move as shown by the         arrows, which allows positioning the 405 nm laser beam (44) to         hit on a certain place as for instance the cantilever (51)     -   42. Hollow scanner system that allows to move the box (31) with         respect to the sample holder (53) in three orthogonal directions         (X, Y and Z). In addition the scanner contains XV positioners to         do a coarse movement of the box (31) with respect to the sample         holder (53)     -   43. Mirror that reflects the 850 nm laser (45) to hit the         photodiode (49)     -   44. 405 nm laser beam     -   45. 850 nm laser beam     -   46. Optical system to correct and focus the 405 nm laser (44)     -   47. Optical system to correct and focus the 850 nm laser (45)     -   48. Band-pass filter center at 850 nm. It allows the 850 nm         laser beam to reach the photodiode (49) but blocks other         wavelengths     -   49. Four quadrant photodiode     -   50. Cantilever holder     -   51. Cantilever chip     -   52. Inverted microscope. It is compatible with modem optical         techniques such as fluorescence, DIC, phase contrast, confocal         microscopy, FRET, Raman spectroscopy     -   53. Sample holder. It contains XY positioners to do a coarse         movement of the sample holder (53) with respect to the box (31)     -   54. Platform where the box (31) is sitting. The platform         contains XY positioner for a coarse movement of the box (31)         with respect to the sample holder (53)     -   55. Hollow scanner system that holds the sample holder (53). It         allows to move and scan the sample holder (53) with respect to         the box (31) in three orthogonal directions (XYZ). In addition         contains XY positioners for a coarse movement of the sample         holder (53) with respect to the box (31)

Having described preferred embodiments of the invention, it will be apparent to those skilled in the art to which this invention relates, that modifications and amendments to various features and items can be effected and yet still come within the general concept of the invention. It is to be understood that all such modifications and amendments are intended to be included within the scope of the present invention. 

1. Art atomic force microscope measuring device comprising a micro-cantilever and an intensity modulated light source exciting the cantilever, wherein the measuring device comprises an optical microscope.
 2. The measuring device according to claim 1, wherein the cantilever is transparent for a wavelength of the visual spectrum.
 3. The measuring device according to claim 1, wherein the cantilever has a length in the range of 10 μm to 1000 μm, and/or a fundamental resonance frequency in a range of 1 Hz to 10 MHz when immersed in water and/or an oscillation amplitude when excited at resonance in the range of 0.01 nm to 500 nm and/or the exciting light has a wavelength in the range of 350 nm to 750 nm.
 4. The measuring device according to claim 1, wherein the exciting light spot is focused on the base of the cantilever.
 5. The measuring device according to claim 1, wherein the exciting light spot is smaller than 100 μm in diameter.
 6. The measuring device according to claim 1, wherein said optical microscope is at least one selected from the group consisting of a fluorescence microscope, a confocal microscope, a fluorescence energy transfer (FRET) microscope, a DIC and/or phase contrast microscope or a Raman spectrometer all of those in particular construed as an inverted microscope.
 7. The measuring device according to claim 3, wherein the cantilever has a length in the size of 10 μm to 500 μm and/or a fundamental resonance frequency in the range of 1 kHz to 2000 kHz, when immersed in water and/or an oscillation amplitude when excited at resonance smaller than 100 nm and/or the exciting light has a wavelength in a range of 350 nm to 450 nm.
 8. The measuring device according to claim 5, wherein the exciting light spot is smaller than 50 μm in diameter in diameter.
 9. The measuring device according to claim 1, wherein the exciting light spot is smaller than 10 μm in diameter. 